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Laboratory Diagnostics
Chapter 17
Laboratory Diagnostics Nicola Bizzaro1,2, Renato Tozzoli1,2 1Laboratory
of Clinical Pathology, Azienda Sanitaria Universitaria Integrata di Udine, San Antonio Hospital, Tolmezzo, Italy; 2Laboratory of Clinical Pathology, Department of Laboratory Medicine, S. Maria degli Angeli Hospital, Pordenone, Italy
Laboratory diagnostics provide two pieces in the mosaic of autoimmunity: the autoantibody tests and the immunological methods used to measure antibodies. Using the most appropriate autoantibody test for each clinical setting and the methods that provide the best diagnostic accuracy for clinical use is a duty of the autoimmunology laboratory. This task has become even more complex today because the scenario has deeply changed since 10–15 years ago: in this time interval, new autoantibody–autoantigen systems (both systemic and organ-specific) have been discovered; new diagnostic technologies have become available and changes in organizational processes have occurred, mainly because of introduction of advanced information technology and test automation [1–3]. Given the wide number of tests and methods currently available and the different roles recognized to many antibodies, the choice of the most appropriate test and method involves organizational and strategic issues that depend on the type of clinical context and the purpose of antibody research [4]. Currently available autoantibody tests contribute significantly to the diagnosis of autoimmune diseases, both for screening purposes when the tests have high sensitivity and for disease classification when the tests are highly specific. However, the search for new antibodies is still needed (1) to find immuno-serological markers in diseases whose autoimmune pathogenesis has only recently been highlighted; (2) to close as far as possible the gap in those diseases in which the already available antibodies display an insufficient diagnostic sensitivity; and (3) to finding antibodies that can be used to monitor therapy. Just to mention only a few of them, new antibody tests have been proposed for deamidated gliadin in celiac disease, carbamylated proteins and 3-14-3η protein in rheumatoid arthritis (RA), anti-PLA2R in primary membranous nephropathy, anti-NMDA receptor in limbic encephalitis, antiamphiphysin and anti-Tr antibodies in paraneoplastic neurological syndromes, anti-beta 2 GPI domain I in antiphospholipid syndrome (APS), antimuscle specific kinase (MuSK) in miastenia gravis, and antiaquaporin 4 antibodies in neuromyelitis optica. Some of these antibodies have been already introduced in clinical practice; for others there is still a need to undertake clinical validation studies before considering routine diagnostic use. In parallel with the new antibodies introduced in the diagnostics, over the years there has been an awareness that antibodies that are already measured daily in many laboratories have characteristics that can be used for different clinical purposes [5]. Here, then, the notion has emerged that antibodies can have not only diagnostic but also prognostic meaning (when they are pathogenetic or involved in the mechanism that causes the disease), can be predictive of disease onset, and ultimately can also be protective, ie., capable to counteract the development of some autoimmune diseases.
THE DIAGNOSTIC AND PROGNOSTIC VALUE OF AUTOANTIBODIES The detection of serum autoantibodies against a wide number of structural and functional molecules present in ubiquitous or tissue-specific cells is used for the diagnosis and classification of autoimmune diseases (Table 17.1). Autoantibodies that are well-recognized criteria of some autoimmune diseases are antinuclear antibodies (ANA), anti-dsDNA, anti-Sm and antiphospholipid antibodies (aPL) for systemic lupus erythematous (SLE), rheumatoid factor for RA, anti-Ro for Sjӧgren’s syndrome, anti-U1RNP for mixed connective tissue disease, antitopoisomerase and anticentromere B protein for systemic sclerosis (SSc), and antimitochondrial antibodies (AMA) for primary biliary cholangitis (PBC). More recently, other antibodies have been included among classification/diagnosis criteria for specific autoimmune disorders, namely anticitrullinated protein antibodies (ACPA) for RA [6], anti-RNA polymerase III antibodies for SSc [7], anti–tissue transglutaminase antibodies (tTG) for celiac disease [8], anti–soluble liver antigen (SLA) for type 1 autoimmune hepatitis [9], and anti-TSH receptor (TRAb) for Graves’ disease [10]. Mosaic of Autoimmunity. https://doi.org/10.1016/B978-0-12-814307-0.00017-7 Copyright © 2019 Elsevier Inc. All rights reserved.
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TABLE 17.1 Diagnostic/Classificative Role of Autoantibodies Antibody
Disease
ANA, anti-dsDNA, anti-Sm, aPL
Systemic lupus erythematosus
Rheumatoid factor
Rheumatoid arthritis
Anti-Ro
Sjӧgren’s syndrome
Anti-U1RNP
Mixed connective tissue disease
Antitopoisomerase, anticentromere B protein
Systemic sclerosis
Antimitochondrial
Primary biliary cholangitis
Anticitrullinated protein
Rheumatoid arthritis
Anti-RNA polymerase III
Systemic sclerosis
Antitissue transglutaminase
Celiac disease
Antisoluble liver antigen
Type 1 autoimmune hepatitis
Anti-TSH receptor
Graves’ disease
TABLE 17.2 Prognostic Role of Autoantibodies Antibody
Disease
Clinical Aspect
Anticitrullinated protein
Rheumatoid arthritis
Clinical progression
Different antibodies
Systemic lupus erythematosus
Nephritis, neonatal lupus, congenital heart block
Antiribosomal P protein
Systemic lupus erythematosus
Cerebritis, psychosis, depression
aPL
Systemic lupus erythematosus
Stroke
aCL, antiprothrombin
Systemic lupus erythematosus
Thrombotic event
Antitopoisomerase I, anticentromere
Systemic sclerosis
Activity and severity
Anti-RNA polymerase III
Systemic sclerosis
Cutaneous thickening, renal crisis
Anti-C1q, anti-dsDNA
Systemic lupus erythematosus
Renal flares
Anti-TSH receptor
Graves’ disease
Remission/recurrence
Anti–alpha actinin
Type 1 autoimmune hepatitis
Response to therapy
Anti-Ro52, soluble liver antigen
Type 1 autoimmune hepatitis
Poor prognosis
In addition, as autoantibodies may reflect the presence, nature, and intensity of the immune response, it is possible to use them as prognostic markers of disease activity and severity in patients who already have been diagnosed with an autoimmune disease (Table 17.2). In RA, the presence of ACPA antibodies is an important independent predictor of the clinical progression [11–16]. In SLE, certain antibodies have been found to be correlated with nephritis, whereas others (i.e., anti-Ro antibodies) were found to represent a significant risk factor for neonatal lupus and congenital heart block in the patient’s sibling. Antiribosomal p protein antibodies have been associated with cerebritis, psychosis, and depression. In addition, aPL have been found to predict stroke in SLE patients [17], and around 50% of SLE patients with anticardiolipin antibodies (aCL) [18] or with antiprothrombin antibodies [19] have a clinical thrombotic event (venous or arterial or recurrent spontaneous abortions) prior to diagnosis.
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Reports have highlighted the close relationship between anti–DNA-topoisomerase I and anticentromere antibody concentration and SSc activity and severity [20]. SSc patients with anti-RNA polymerase III antibodies have the most severe cutaneous thickening and the highest frequency of renal crisis [21]. It has been shown that active nephritis in SLE is associated with higher concentrations of anti-C1q antibodies and that this type of autoantibody is a predictor of renal flares with a specificity superior or similar to that of anti-dsDNA antibodies [22,23]. Antineutrophil cytoplasmic antibodies (ANCA) are considered sensitive markers of disease activity in patients with small-vessel vasculitides, with a moderate ability to predict relapse and guide therapeutic decisions [24,25]. Antibody prediction in prognosis is not restricted to rheumatic diseases but extends to organ-specific autoimmune diseases. In patients with Graves’ disease, TRAb can predict remission or recurrence in patients undergoing antithyroid drug treatment [26]; antismooth muscle antibodies predict development of autoimmune hepatitis in patients with normal liver function [27], and anti–alpha actinin antibodies may predict response to therapy in patients with type 1 autoimmune hepatitis [28]. In addition, anti-Ro52 antibodies associated to SLA antibodies indicate a poor prognosis in type 1 autoimmune hepatitis [29].
THE PREDICTIVE VALUE OF AUTOANTIBODIES Besides their diagnostic and prognostic role, many specific autoantibodies are early screening indicators, as they can be detected in the sera of asymptomatic subjects who later eventually develop overt systemic [30–32] or organ-specific [33,34] autoimmune diseases (Table 17.3). Studies highlighting the predictive value of autoantibodies are important because they redefined the natural history of autoimmune diseases as a group of human diseases characterized by a long latency period and by the appearance of autoantibodies when clinical manifestations are still absent. Among autoimmune rheumatic diseases, antibodies to U1RNP, Sm, dsDNA, cardiolipin, Ro, and La have a positive predictive value (PPV) for SLE ranging from 94% to 100%. According to the type of antibody, the appearance can precede clinical diagnosis by 7–10 years with a frequency that varies from 32% to 78% at the moment of diagnosis [35]. Moderate or high levels of aCL are present in 18% of asymptomatic individuals up to 7 years before SLE diagnosis [18]. In subjects with scleroderma, anticentromere and antitopoisomerase I antibodies are detectable up to 11 years before clinical manifestations, with a PPV of 100%. In RA, rheumatoid factor has a predictivity between 52% and 88%, whereas for ACPA the predictivity is much higher, reaching 97%. These two antibodies have been detected in serum up to 14 years before patients manifest the first symptoms of the disease [36,37]. Anti-Ro and anti-La antibodies have been detected on average 5 years before diagnosis in 73% of asymptomatic mothers who had given birth to a child with autoantibody-associated congenital heart block and who later developed Sjögren’s syndrome [38,39]. Antisynthetase antibodies may be found in patients with idiopathic inflammatory myositis years before disease onset [40], and antinucleosome antibodies were found to be present in 67% of patients with APS up to 11 years before the development of SLE [41].
TABLE 17.3 Predictive Value of Autoantibodies Antibody
Disease
Anti-U1RNP, anti-dsDNA, anti-Sm, aCL, anti-Ro, anti-La
Systemic lupus erythematosus
Rheumatoid factor, citrullinated protein
Rheumatoid arthritis
Anti-Ro, anti-La
Sjӧgren’s syndrome
Anti-TPO
Postpartum thyroiditis
Parietal cells
Autoimmune gastritis
Antimitochondrial
Primary biliary cholangitis
Anti-insulin, anti-GAD, anti-IA-2, anti-zinc transporter 8
Type 1 diabetes mellitus
Adrenal cortex
Addison’s disease
Anti–Saccharomyces cerevisiae
Crohn’s disease
Antitissue transglutaminase, antiendomysial
Celiac disease
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In organ-specific autoimmune diseases, the predictive value of each antibody characteristic for a specific disease is similar to that found for the autoantibodies in autoimmune rheumatic diseases. Antithyroid peroxidase antibodies (anti-TPO) have been shown to be a good predictor of postpartum thyroid dysfunction. A high anti-TPO antibody level immediately postpartum can predict thyroiditis with 97% sensitivity, 91% specificity, and a PPV of 92% [42]. The presence of antiparietal cell antibodies predicts the development of autoimmune gastritis [43]. In AMA-positive subjects without clinical or biochemical signs of hepatic damage, AMA can be detected up to 25 years before the clinical manifestation of PBC [44]. Their PPV is higher of 95% [45]. Pancreatic islet cells antibodies and antibodies to insulin, to 65-kD glutamic acid decarboxylase (GAD), and to tyrosine phosphatase-like (IA-2) protein are predictive markers of type 1 diabetes mellitus (T1DM) [46]. Their PPV is 43%, 55%, 42%, and 29%, respectively [47]. The risk of developing the disease in first-degree relatives of patients with diabetes grows progressively with the duration of the follow-up and with the number of positive autoantibodies, being 2%, 25%, and 70%, with one, two, and three or four positive antibodies [47]. Recently, antibodies to the zinc transporter 8 (ZnT8) were shown to predict diabetes independently of antiislet cell antibodies [48] and antibodies to GAD could predict development of thyroid autoimmune diseases in adult patients with T1DM [49]. Adrenal cortex autoantibodies may precede Addison’ s disease onset by up to 10 years and their PPV is about 70% [50]. Anti–Saccharomyces cerevisiae antibodies as markers for Crohn’s disease have been detected in the sera of apparently healthy subjects, on average 3 years before the disease became manifest. Their diagnostic sensitivity was 31%, but specificity and predictive value were both 100% [51]. The predictive value for celiac disease onset of anti-tTG and antiendomysial antibodies is 50%–60%. If the patient carries the HLA-DQ2 or DQ8 antigens, known to be genetic markers for celiac disease susceptibility, the PPV of autoantibodies approaches 100% [52].
THE PROTECTIVE ROLE OF AUTOANTIBODIES Natural polyreactive autoantibodies, mostly of the IgM isotype, are found in the sera of healthy individuals and react with both self and nonself antigens. Nonspecific and low-affinity binding of these natural autoantibodies to self-antigens may prevent autoreactive clones from reacting with self-antigens by masking their antigenic determinants [53]. Protective autoantibodies are thought to play a role in preventing many autoimmune diseases, such as SLE. Indeed, IgM anti-dsDNA antibodies were found to be significantly associated with milder disease activity and have a negative correlation with the severity of glomerulonephritis in SLE [54,55]; in addition, their administration into SLE-prone mice prevented the development of nephritis. Interestingly, the presence of antinuclear and anti-dsDNA antibodies is associated with a better prognosis of cancer suggesting that these autoantibodies may function as potential antineoplastic agents [56]. IgM antibodies to oxidized low density lipoproteins (LDL) have been suggested to prevent the clinical manifestations of atherosclerosis [57] and the presence of rheumatoid factor in SLE to be protective against the development of lupus nephritis.
THE METHODS TO DETECT AUTOANTIBODIES Compared with the methods used 10 years ago, the mosaic has further expanded. Although, for the time being, the method of indirect immunofluorescence (IIF) maintains its basic role in autoimmune diagnostics, to cope with increased antibody request and at the same time continue to exploit the high sensitivity of IIF, automated systems have been developed for reading and interpretation of IIF tests for ANA. Although their performance needs to be further improved, these systems already allow for automated classification of samples, with a high efficiency in discriminating between positive and negative ANA and an acceptable correlation with manual microscope reading [58]. Automated quantification of autoantibodies [59,60] and implementation of a quantitative internal QC system [61] are possible further advantageous applications of this IIF method innovation. In addition, the availability of these systems suggests that automation of cell-based IIF testing may improve standardization of antibody testing and help to reduce variability among autoimmunology laboratories. Automated systems have been developed also for the detection of anti-dsDNA antibodies on Crithidia luciliae substrate [62] and for ANCA [63,64]. Efforts are in progress to extend this technology to tissues for the detection of antibodies to gastric parietal cells, smooth muscle, mitochondrial, and endomysial antigens. Besides these recent improvements in IIF, a wide number of different immunoassays (monoplex and multiplex) have been introduced and are currently used for single or multiple measurements of autoantibodies [65]. A classification of these methods is shown in Table 17.4. While immunoenzymatic methods (ELISA) are progressively abandoned in clinical laboratories, fully automated random access chemiluminescence (CLIA) instruments are emerging as a promising technology and are destined for further development [66]. Current chemiluminescent immunoassays consist of tests that measure one autoantibody at a time.
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TABLE 17.4 Classification of Immunoassay Methods for the Detection of Autoantibodies First-Generation Monoplex
Abbreviation
Double immunodiffusion
ID
Complement fixation
CF
Indirect immunofluorescence
IIF
Passive agglutination
PHA
Radioimmunoprecipitation
RIPA
Western blot/immunodot
WB/IB
Second–Third Generation Monoplex
Abbreviation
Radioimmunoassay–immunoradiometric assay
RIA-IRMA
Radioreceptor assay
RRA
Immunoenzymatic assay–immunoenzymometric assay
ELISA-IEMA
Immunoblot
IB
Immunodot
DB
Chemiluminescence immunoassay
CLIA-ILMA
Fluorescence immunoassay
FIA-IFMA
Multiplex
Abbreviation
Nonplanar (addressable microbeads) immunoassay
NPMIA
Planar (membranes, glass slides) immunoassay
PMIA
However, the need is emerging for multiparametric tests that can identify all the components of the immunological picture in a single analytical step, efficiently and at reasonable cost. Use of the two-dimensional resolution for CL multiplex immunoassay [67,68] and of the ultrasensitive chemiluminescence magnetic nanoparticles immunoassay technology will further increase the analytic sensitivity of the CLIA method [69,70] and might open doors for the setting up of multiparametric CLIA tests enabling significantly reduction in the analysis time [71]. Also, planar and nonplanar autoantigenic arrays have found application for research on autoantibody profiling of autoimmune diseases [72–74]. Planar array systems are made up of microspots on glass slides or on polystyrene microplates and linear immunoblot systems on nitrocellulose membranes. Among the nonplanar arrays there have been developed systems in suspension that use microparticles recognized by laser nephelometry or laser fluorimetry in flow cytometry [75]. Many of these systems are already used in diagnostics; others are still in the early stages of development and need clinical validation, but their ease of use and speed of analysis suggest them as valid alternatives to current immunometric methods.
ORGANIZATION OF THE AUTOIMMUNE LABORATORY The progressive increase in requests of autoantibody tests that occurred in recent years has brought to the introduction of subtotal and total automation in the clinical immunology laboratory [76,77]. Currently, all stages of the analytical procedure for detection and quantification of autoantibodies are automated. The third generation of laboratory systems now encompasses most of the analytical steps of the laboratory workflow, enabling the clinical pathologists to focus on “valueadded” work, such as result validation and production of narrative reports for clinical interpretation [3]. Automation technology has many advantages: it can reduce labor requirements; improve turnaround time; achieve higher quality of testing (precision, limits of detections, dynamic range, etc.); reduce pre-, post-, and analytical errors; and increase throughput and productivity. The choice of the best strategy depends on many factors: the tests requested (screening, diagnostic confirmation, monitoring); the pathology to be investigated (rheumatic disease, liver disease, celiac disease, etc.); technologies available to the laboratory; diagnostic accuracy; and cost of test and methods.
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A final note concerns the possibility that new technologies offer today to respond in a very short time to the request of autoantibody tests. Although in most cases this is not clinically justified, real-time measurement of autoantibodies may clearly benefit the rheumatology or the nephrology practice in emergency and urgent care settings. Furthermore, the availability of automated analyzers with reduced assay times as well of manual point-of-care systems enables real-time antibody measurement in the same day of the request or even in stat mode, avoiding delay and improving compliance [78]. This also responds to the increasing need for faster diagnosis owing to shorter period of hospitalization. In conclusion, in recent years major changes have occurred in the laboratory diagnostics of autoimmune diseases. Physicians need to fully understand these changes for appropriate test requests; the laboratory autoimmunologist needs to acquire a higher clinical expertise and a complete knowledge of the pros and cons of new analytical methods, far superior to the one required only a few years ago, for optimal clinical governance.
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[58] Bizzaro N, Antico A, Platzgummer S, Tonutti E, Bassetti D, Pesente F, et al. Automated antinuclear immunofluorescence antibody screening: a comparative study of six computer-aided diagnostic systems. Autoimmun Rev 2013;13:292–8. [59] Bertin D, Jourde-Chiche N, Bongrand P, Bardin N. Original approach for automated quantification of antinuclear autoantibodies by indirect immunofluorescence. Clin Dev Immunol 2013:182172. [60] Peng X, Tang J, Wu Y, Yang B, Hu J. Novel method for ANA quantitation using IIF imaging system. J Immunol Methods 2014;404:52–8. [61] Maenhout TM, Bonroy C, Verfaillie SV, Devreese K. Automated indirect immunofluorescence microscopy enables the implementation of a quantitative internal quality control system for anti-nuclear antibody (ANA) analysis. Clin Chem Lab Med 2014;52:989–98. [62] Buzzulini F, Rigon A, Soda P, Onofri L, Infantino M, Arcarese L, et al. The classification of Crithidia luciliae immunofluorescence test (CLIFT) using a novel automated system. Arthritis Res Ther 2014;16:R71. [63] Knütter I, Hiemann R, Brumma T, Büttner T, Großmann K, Cusini M. Automated interpretation of ANCA patterns a new approach in the serology of ANCAassociated vasculitis. Arthritis Res Ther 2012;14:R271. [64] Sowa M, Grossmann K, Knutter I, Hiemann R, Rober N, Anderer U, et al. Simultaneous automated screening and confirmatory testing for vasculitisspecific ANCA. PLoS One 2014;9. e107743. [65] Tozzoli R, Sorrentino MC, Bizzaro N. Detecting multiple autoantibodies to diagnose autoimmune comorbidity (multiple autoimmune syndromes and overlap syndromes): a challenge for the autoimmunologist. Immunol Res 2013;56:425–31. [66] Cinquanta L, Fontana DE, Bizzaro N. Chemiluminescent immunoassay technology: what does it change in autoantibody detection? Autoimmun Highlights 2017;8:9. [67] Fu ZF, Liu H, Ju HX. Flow-through multianalyte chemiluminescent immunosensing system with designed substrate zone-resolved technique for sequential detection of tumor markers. Anal Chem 2006;78:6999–7005. [68] Liu H, Fu ZF, Yang ZJ, Yan F, Ju HX. Sampling-resolution strategy for one-way multiplexed immunoassay with sequential chemiluminescent detection. Anal Chem 2008;80:5654–9. [69] Du L, Ji W, Zhang Y, Zhang C, Liu G, Wang S. An ultrasensitive detection of 17β-estradiol using a gold nanoparticle-based fluorescence immunoassay. Analyst 2015;140:2001–7. [70] Tao X, Jiang H, Yu X, Zhu J, Wang X, Wang Z, et al. An ultrasensitive chemiluminescence immunoassay of chloramphenicol based on gold nanoparticles and magnetic beads. Drug Test Anal 2013;53:46–52. [71] Wang C, Wu J, Zong C, Xu J, Ju HX. Chemiluminescent immunoassay and its applications. Chin J Anal Chem 2012;40:3–10. [72] Tozzoli R, Bizzaro N. Novel diagnostic methods for autoantibody detection. In: Shoenfeld Y, Gershwin ME, Meroni PL, editors. Autoantibodies. Amsterdam: Elsevier; 2006. p. 77–82. [73] Villalta D, Girolami E, Alessio MG, Sorrentino MC, Tampoia M, Brusca I, et al. Autoantibody profiling in a cohort of pediatric and adult patients with autoimmune hepatitis. J Clin Lab Anal 2016;30:41–6. [74] Villalta D, Sorrentino MC, Girolami E, Tampoia M, Alessio MG, Brusca I, et al. Autoantibody profiling of patients with primary biliary cirrhosis using a multiplexed line-blot assay. Chim Clin Acta 2015;438:135–8. [75] Fulton RJ, McDade RL, Smith PL, Kienker LJ, Kettman JRJ. Advanced multiplexed analysis with the FluoMetrix system. Clin Chem 1997;43:1749–56. [76] Armbruster DA, Overcash DR, Reyes J. Clinical chemistry laboratory automation in the 21st century – Amat Victoria curam (victory loves careful preparation). Clin Biochem Rev 2014;35:143–53. [77] Tozzoli R, D’Aurizio F, Villalta D, Bizzaro N. Automation, consolidation, and integration in autoimmune diagnostics. Autoimmun Highlights July 3, 2015. https://doi.org/10.1007/s13317-015-0067-5. [78] Kostantinov KN, Tzamaloukas A, Rubin RL. Detection of autoantibodies in a point-of-care rheumatology setting. Autoimmun Highlights 2013;4:55–61.
Laboratory Diagnostics Nicola Bizzaro1,2, Renato Tozzoli1,2 1Laboratory
of Clinical Pathology, Azienda Sanitaria Universitaria Integrata di Udine, San Antonio Hospital, Tolmezzo, Italy; 2Laboratory of Clinical Pathology, Department of Laboratory Medicine, S. Maria degli Angeli Hospital, Pordenone, Italy
Laboratory diagnostics provide two pieces in the mosaic of autoimmunity: the autoantibody tests and the immunological methods used to measure antibodies. Using the most appropriate autoantibody test for each clinical setting and the methods that provide the best diagnostic accuracy for clinical use is a duty of the autoimmunology laboratory. This task has become even more complex today because the scenario has deeply changed since 10–15 years ago: in this time interval, new autoantibody–autoantigen systems (both systemic and organ-specific) have been discovered; new diagnostic technologies have become available and changes in organizational processes have occurred, mainly because of introduction of advanced information technology and test automation [1–3]. Given the wide number of tests and methods currently available and the different roles recognized to many antibodies, the choice of the most appropriate test and method involves organizational and strategic issues that depend on the type of clinical context and the purpose of antibody research [4]. Currently available autoantibody tests contribute significantly to the diagnosis of autoimmune diseases, both for screening purposes when the tests have high sensitivity and for disease classification when the tests are highly specific. However, the search for new antibodies is still needed (1) to find immuno-serological markers in diseases whose autoimmune pathogenesis has only recently been highlighted; (2) to close as far as possible the gap in those diseases in which the already available antibodies display an insufficient diagnostic sensitivity; and (3) to finding antibodies that can be used to monitor therapy. Just to mention only a few of them, new antibody tests have been proposed for deamidated gliadin in celiac disease, carbamylated proteins and 3-14-3η protein in rheumatoid arthritis (RA), anti-PLA2R in primary membranous nephropathy, anti-NMDA receptor in limbic encephalitis, antiamphiphysin and anti-Tr antibodies in paraneoplastic neurological syndromes, anti-beta 2 GPI domain I in antiphospholipid syndrome (APS), antimuscle specific kinase (MuSK) in miastenia gravis, and antiaquaporin 4 antibodies in neuromyelitis optica. Some of these antibodies have been already introduced in clinical practice; for others there is still a need to undertake clinical validation studies before considering routine diagnostic use. In parallel with the new antibodies introduced in the diagnostics, over the years there has been an awareness that antibodies that are already measured daily in many laboratories have characteristics that can be used for different clinical purposes [5]. Here, then, the notion has emerged that antibodies can have not only diagnostic but also prognostic meaning (when they are pathogenetic or involved in the mechanism that causes the disease), can be predictive of disease onset, and ultimately can also be protective, ie., capable to counteract the development of some autoimmune diseases.
THE DIAGNOSTIC AND PROGNOSTIC VALUE OF AUTOANTIBODIES The detection of serum autoantibodies against a wide number of structural and functional molecules present in ubiquitous or tissue-specific cells is used for the diagnosis and classification of autoimmune diseases (Table 17.1). Autoantibodies that are well-recognized criteria of some autoimmune diseases are antinuclear antibodies (ANA), anti-dsDNA, anti-Sm and antiphospholipid antibodies (aPL) for systemic lupus erythematous (SLE), rheumatoid factor for RA, anti-Ro for Sjӧgren’s syndrome, anti-U1RNP for mixed connective tissue disease, antitopoisomerase and anticentromere B protein for systemic sclerosis (SSc), and antimitochondrial antibodies (AMA) for primary biliary cholangitis (PBC). More recently, other antibodies have been included among classification/diagnosis criteria for specific autoimmune disorders, namely anticitrullinated protein antibodies (ACPA) for RA [6], anti-RNA polymerase III antibodies for SSc [7], anti–tissue transglutaminase antibodies (tTG) for celiac disease [8], anti–soluble liver antigen (SLA) for type 1 autoimmune hepatitis [9], and anti-TSH receptor (TRAb) for Graves’ disease [10]. Mosaic of Autoimmunity. https://doi.org/10.1016/B978-0-12-814307-0.00017-7 Copyright © 2019 Elsevier Inc. All rights reserved.
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TABLE 17.1 Diagnostic/Classificative Role of Autoantibodies Antibody
Disease
ANA, anti-dsDNA, anti-Sm, aPL
Systemic lupus erythematosus
Rheumatoid factor
Rheumatoid arthritis
Anti-Ro
Sjӧgren’s syndrome
Anti-U1RNP
Mixed connective tissue disease
Antitopoisomerase, anticentromere B protein
Systemic sclerosis
Antimitochondrial
Primary biliary cholangitis
Anticitrullinated protein
Rheumatoid arthritis
Anti-RNA polymerase III
Systemic sclerosis
Antitissue transglutaminase
Celiac disease
Antisoluble liver antigen
Type 1 autoimmune hepatitis
Anti-TSH receptor
Graves’ disease
TABLE 17.2 Prognostic Role of Autoantibodies Antibody
Disease
Clinical Aspect
Anticitrullinated protein
Rheumatoid arthritis
Clinical progression
Different antibodies
Systemic lupus erythematosus
Nephritis, neonatal lupus, congenital heart block
Antiribosomal P protein
Systemic lupus erythematosus
Cerebritis, psychosis, depression
aPL
Systemic lupus erythematosus
Stroke
aCL, antiprothrombin
Systemic lupus erythematosus
Thrombotic event
Antitopoisomerase I, anticentromere
Systemic sclerosis
Activity and severity
Anti-RNA polymerase III
Systemic sclerosis
Cutaneous thickening, renal crisis
Anti-C1q, anti-dsDNA
Systemic lupus erythematosus
Renal flares
Anti-TSH receptor
Graves’ disease
Remission/recurrence
Anti–alpha actinin
Type 1 autoimmune hepatitis
Response to therapy
Anti-Ro52, soluble liver antigen
Type 1 autoimmune hepatitis
Poor prognosis
In addition, as autoantibodies may reflect the presence, nature, and intensity of the immune response, it is possible to use them as prognostic markers of disease activity and severity in patients who already have been diagnosed with an autoimmune disease (Table 17.2). In RA, the presence of ACPA antibodies is an important independent predictor of the clinical progression [11–16]. In SLE, certain antibodies have been found to be correlated with nephritis, whereas others (i.e., anti-Ro antibodies) were found to represent a significant risk factor for neonatal lupus and congenital heart block in the patient’s sibling. Antiribosomal p protein antibodies have been associated with cerebritis, psychosis, and depression. In addition, aPL have been found to predict stroke in SLE patients [17], and around 50% of SLE patients with anticardiolipin antibodies (aCL) [18] or with antiprothrombin antibodies [19] have a clinical thrombotic event (venous or arterial or recurrent spontaneous abortions) prior to diagnosis.
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Reports have highlighted the close relationship between anti–DNA-topoisomerase I and anticentromere antibody concentration and SSc activity and severity [20]. SSc patients with anti-RNA polymerase III antibodies have the most severe cutaneous thickening and the highest frequency of renal crisis [21]. It has been shown that active nephritis in SLE is associated with higher concentrations of anti-C1q antibodies and that this type of autoantibody is a predictor of renal flares with a specificity superior or similar to that of anti-dsDNA antibodies [22,23]. Antineutrophil cytoplasmic antibodies (ANCA) are considered sensitive markers of disease activity in patients with small-vessel vasculitides, with a moderate ability to predict relapse and guide therapeutic decisions [24,25]. Antibody prediction in prognosis is not restricted to rheumatic diseases but extends to organ-specific autoimmune diseases. In patients with Graves’ disease, TRAb can predict remission or recurrence in patients undergoing antithyroid drug treatment [26]; antismooth muscle antibodies predict development of autoimmune hepatitis in patients with normal liver function [27], and anti–alpha actinin antibodies may predict response to therapy in patients with type 1 autoimmune hepatitis [28]. In addition, anti-Ro52 antibodies associated to SLA antibodies indicate a poor prognosis in type 1 autoimmune hepatitis [29].
THE PREDICTIVE VALUE OF AUTOANTIBODIES Besides their diagnostic and prognostic role, many specific autoantibodies are early screening indicators, as they can be detected in the sera of asymptomatic subjects who later eventually develop overt systemic [30–32] or organ-specific [33,34] autoimmune diseases (Table 17.3). Studies highlighting the predictive value of autoantibodies are important because they redefined the natural history of autoimmune diseases as a group of human diseases characterized by a long latency period and by the appearance of autoantibodies when clinical manifestations are still absent. Among autoimmune rheumatic diseases, antibodies to U1RNP, Sm, dsDNA, cardiolipin, Ro, and La have a positive predictive value (PPV) for SLE ranging from 94% to 100%. According to the type of antibody, the appearance can precede clinical diagnosis by 7–10 years with a frequency that varies from 32% to 78% at the moment of diagnosis [35]. Moderate or high levels of aCL are present in 18% of asymptomatic individuals up to 7 years before SLE diagnosis [18]. In subjects with scleroderma, anticentromere and antitopoisomerase I antibodies are detectable up to 11 years before clinical manifestations, with a PPV of 100%. In RA, rheumatoid factor has a predictivity between 52% and 88%, whereas for ACPA the predictivity is much higher, reaching 97%. These two antibodies have been detected in serum up to 14 years before patients manifest the first symptoms of the disease [36,37]. Anti-Ro and anti-La antibodies have been detected on average 5 years before diagnosis in 73% of asymptomatic mothers who had given birth to a child with autoantibody-associated congenital heart block and who later developed Sjögren’s syndrome [38,39]. Antisynthetase antibodies may be found in patients with idiopathic inflammatory myositis years before disease onset [40], and antinucleosome antibodies were found to be present in 67% of patients with APS up to 11 years before the development of SLE [41].
TABLE 17.3 Predictive Value of Autoantibodies Antibody
Disease
Anti-U1RNP, anti-dsDNA, anti-Sm, aCL, anti-Ro, anti-La
Systemic lupus erythematosus
Rheumatoid factor, citrullinated protein
Rheumatoid arthritis
Anti-Ro, anti-La
Sjӧgren’s syndrome
Anti-TPO
Postpartum thyroiditis
Parietal cells
Autoimmune gastritis
Antimitochondrial
Primary biliary cholangitis
Anti-insulin, anti-GAD, anti-IA-2, anti-zinc transporter 8
Type 1 diabetes mellitus
Adrenal cortex
Addison’s disease
Anti–Saccharomyces cerevisiae
Crohn’s disease
Antitissue transglutaminase, antiendomysial
Celiac disease
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In organ-specific autoimmune diseases, the predictive value of each antibody characteristic for a specific disease is similar to that found for the autoantibodies in autoimmune rheumatic diseases. Antithyroid peroxidase antibodies (anti-TPO) have been shown to be a good predictor of postpartum thyroid dysfunction. A high anti-TPO antibody level immediately postpartum can predict thyroiditis with 97% sensitivity, 91% specificity, and a PPV of 92% [42]. The presence of antiparietal cell antibodies predicts the development of autoimmune gastritis [43]. In AMA-positive subjects without clinical or biochemical signs of hepatic damage, AMA can be detected up to 25 years before the clinical manifestation of PBC [44]. Their PPV is higher of 95% [45]. Pancreatic islet cells antibodies and antibodies to insulin, to 65-kD glutamic acid decarboxylase (GAD), and to tyrosine phosphatase-like (IA-2) protein are predictive markers of type 1 diabetes mellitus (T1DM) [46]. Their PPV is 43%, 55%, 42%, and 29%, respectively [47]. The risk of developing the disease in first-degree relatives of patients with diabetes grows progressively with the duration of the follow-up and with the number of positive autoantibodies, being 2%, 25%, and 70%, with one, two, and three or four positive antibodies [47]. Recently, antibodies to the zinc transporter 8 (ZnT8) were shown to predict diabetes independently of antiislet cell antibodies [48] and antibodies to GAD could predict development of thyroid autoimmune diseases in adult patients with T1DM [49]. Adrenal cortex autoantibodies may precede Addison’ s disease onset by up to 10 years and their PPV is about 70% [50]. Anti–Saccharomyces cerevisiae antibodies as markers for Crohn’s disease have been detected in the sera of apparently healthy subjects, on average 3 years before the disease became manifest. Their diagnostic sensitivity was 31%, but specificity and predictive value were both 100% [51]. The predictive value for celiac disease onset of anti-tTG and antiendomysial antibodies is 50%–60%. If the patient carries the HLA-DQ2 or DQ8 antigens, known to be genetic markers for celiac disease susceptibility, the PPV of autoantibodies approaches 100% [52].
THE PROTECTIVE ROLE OF AUTOANTIBODIES Natural polyreactive autoantibodies, mostly of the IgM isotype, are found in the sera of healthy individuals and react with both self and nonself antigens. Nonspecific and low-affinity binding of these natural autoantibodies to self-antigens may prevent autoreactive clones from reacting with self-antigens by masking their antigenic determinants [53]. Protective autoantibodies are thought to play a role in preventing many autoimmune diseases, such as SLE. Indeed, IgM anti-dsDNA antibodies were found to be significantly associated with milder disease activity and have a negative correlation with the severity of glomerulonephritis in SLE [54,55]; in addition, their administration into SLE-prone mice prevented the development of nephritis. Interestingly, the presence of antinuclear and anti-dsDNA antibodies is associated with a better prognosis of cancer suggesting that these autoantibodies may function as potential antineoplastic agents [56]. IgM antibodies to oxidized low density lipoproteins (LDL) have been suggested to prevent the clinical manifestations of atherosclerosis [57] and the presence of rheumatoid factor in SLE to be protective against the development of lupus nephritis.
THE METHODS TO DETECT AUTOANTIBODIES Compared with the methods used 10 years ago, the mosaic has further expanded. Although, for the time being, the method of indirect immunofluorescence (IIF) maintains its basic role in autoimmune diagnostics, to cope with increased antibody request and at the same time continue to exploit the high sensitivity of IIF, automated systems have been developed for reading and interpretation of IIF tests for ANA. Although their performance needs to be further improved, these systems already allow for automated classification of samples, with a high efficiency in discriminating between positive and negative ANA and an acceptable correlation with manual microscope reading [58]. Automated quantification of autoantibodies [59,60] and implementation of a quantitative internal QC system [61] are possible further advantageous applications of this IIF method innovation. In addition, the availability of these systems suggests that automation of cell-based IIF testing may improve standardization of antibody testing and help to reduce variability among autoimmunology laboratories. Automated systems have been developed also for the detection of anti-dsDNA antibodies on Crithidia luciliae substrate [62] and for ANCA [63,64]. Efforts are in progress to extend this technology to tissues for the detection of antibodies to gastric parietal cells, smooth muscle, mitochondrial, and endomysial antigens. Besides these recent improvements in IIF, a wide number of different immunoassays (monoplex and multiplex) have been introduced and are currently used for single or multiple measurements of autoantibodies [65]. A classification of these methods is shown in Table 17.4. While immunoenzymatic methods (ELISA) are progressively abandoned in clinical laboratories, fully automated random access chemiluminescence (CLIA) instruments are emerging as a promising technology and are destined for further development [66]. Current chemiluminescent immunoassays consist of tests that measure one autoantibody at a time.
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TABLE 17.4 Classification of Immunoassay Methods for the Detection of Autoantibodies First-Generation Monoplex
Abbreviation
Double immunodiffusion
ID
Complement fixation
CF
Indirect immunofluorescence
IIF
Passive agglutination
PHA
Radioimmunoprecipitation
RIPA
Western blot/immunodot
WB/IB
Second–Third Generation Monoplex
Abbreviation
Radioimmunoassay–immunoradiometric assay
RIA-IRMA
Radioreceptor assay
RRA
Immunoenzymatic assay–immunoenzymometric assay
ELISA-IEMA
Immunoblot
IB
Immunodot
DB
Chemiluminescence immunoassay
CLIA-ILMA
Fluorescence immunoassay
FIA-IFMA
Multiplex
Abbreviation
Nonplanar (addressable microbeads) immunoassay
NPMIA
Planar (membranes, glass slides) immunoassay
PMIA
However, the need is emerging for multiparametric tests that can identify all the components of the immunological picture in a single analytical step, efficiently and at reasonable cost. Use of the two-dimensional resolution for CL multiplex immunoassay [67,68] and of the ultrasensitive chemiluminescence magnetic nanoparticles immunoassay technology will further increase the analytic sensitivity of the CLIA method [69,70] and might open doors for the setting up of multiparametric CLIA tests enabling significantly reduction in the analysis time [71]. Also, planar and nonplanar autoantigenic arrays have found application for research on autoantibody profiling of autoimmune diseases [72–74]. Planar array systems are made up of microspots on glass slides or on polystyrene microplates and linear immunoblot systems on nitrocellulose membranes. Among the nonplanar arrays there have been developed systems in suspension that use microparticles recognized by laser nephelometry or laser fluorimetry in flow cytometry [75]. Many of these systems are already used in diagnostics; others are still in the early stages of development and need clinical validation, but their ease of use and speed of analysis suggest them as valid alternatives to current immunometric methods.
ORGANIZATION OF THE AUTOIMMUNE LABORATORY The progressive increase in requests of autoantibody tests that occurred in recent years has brought to the introduction of subtotal and total automation in the clinical immunology laboratory [76,77]. Currently, all stages of the analytical procedure for detection and quantification of autoantibodies are automated. The third generation of laboratory systems now encompasses most of the analytical steps of the laboratory workflow, enabling the clinical pathologists to focus on “valueadded” work, such as result validation and production of narrative reports for clinical interpretation [3]. Automation technology has many advantages: it can reduce labor requirements; improve turnaround time; achieve higher quality of testing (precision, limits of detections, dynamic range, etc.); reduce pre-, post-, and analytical errors; and increase throughput and productivity. The choice of the best strategy depends on many factors: the tests requested (screening, diagnostic confirmation, monitoring); the pathology to be investigated (rheumatic disease, liver disease, celiac disease, etc.); technologies available to the laboratory; diagnostic accuracy; and cost of test and methods.
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A final note concerns the possibility that new technologies offer today to respond in a very short time to the request of autoantibody tests. Although in most cases this is not clinically justified, real-time measurement of autoantibodies may clearly benefit the rheumatology or the nephrology practice in emergency and urgent care settings. Furthermore, the availability of automated analyzers with reduced assay times as well of manual point-of-care systems enables real-time antibody measurement in the same day of the request or even in stat mode, avoiding delay and improving compliance [78]. This also responds to the increasing need for faster diagnosis owing to shorter period of hospitalization. In conclusion, in recent years major changes have occurred in the laboratory diagnostics of autoimmune diseases. Physicians need to fully understand these changes for appropriate test requests; the laboratory autoimmunologist needs to acquire a higher clinical expertise and a complete knowledge of the pros and cons of new analytical methods, far superior to the one required only a few years ago, for optimal clinical governance.
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